Biomedical subjects

R Bhushan

Publications and source records attributed to R Bhushan.

At least 19 recordsLinked to original sources

PubMed2001

Level of maternal antibody required to protect neonates against early-onset disease caused by group B Streptococcus type Ia: a multicenter, seroepidemiology study.

Because of the difficulty of conducting efficacy trials of vaccines against group B streptococcus (GBS), the licensure of these vaccines may have to rely on studies that measure vaccine-induced antibody levels that correlate with protection. This study estimates the level of maternal antibody required to protect neonates against early-onset disease (EOD) caused by GBS type Ia. Levels of maternal serum IgG GBS Ia antibodies, measured by ELISAs in 45 case patients (neonates with EOD caused by GBS Ia) and in 319 control subjects (neonates colonized by GBS Ia but without EOD) born at > or =34 weeks gestation were compared. The probability of developing EOD declined with increasing maternal levels of IgG GBS Ia antibody (P = .03). Neonates whose mothers had levels of IgG GBS Ia antibody > or =5 microg/mL had an 88% lower risk (95% confidence interval, 7%-98%) of developing type-specific EOD, compared with those whose mothers had levels < 0.5 microg/mL. A vaccine that induces IgG GBS Ia antibody levels > or =5 microg/mL in mothers can be predicted to confer a high degree of type-specific immunity to EOD to their infants.

Age of Onset↗

PubMed2001

Chiral resolution.

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Chromatography↗

PubMed2001

Direct resolution of (+/-)-ephedrine and atropine into their enantiomers by impregnated TLC.

Direct resolution of (+/-)-ephedrine and atropine into their enantiomers was achieved by normal-phase thin layer chromatography on silica gel plates impregnated with optically pure L-tartaric acid and L-histidine, respectively, as chiral selectors. The mobile phases enabling successful resolution were different combinations of acetonitrile-methanol-water. The spots were detected with iodine vapours and the detection limits were 2 and 6 microg, respectively, in terms of the racemate. The effects of concentration of the impregnating reagent, temperature and pH on resolution have been studied.

Acetonitriles↗

PubMed2001

Separation of amino acids, their derivatives and enantiomers by impregnated TLC.

The present state of TLC with respect to separation of amino acids, their different derivatives and their enantiomers by the technique of impregnation is discussed. The main approaches to impregnation viz. mixing of a suitable reagent with the adsorbent prior to plate-making, immersion of the untreated plate in the solution of impregnating reagent prior to development, and modification of the adsorbent, have been identified and discussed for each class of these compounds. The role of impregnation in resolving enantiomers or in improving the separation of mixtures of amino acids or their derivatives in terms of ion pairing, complex formation, ligand exchange or other steric interactions has been elaborated in each category.

Adsorption↗

PubMed2000

Direct thin layer chromatography enantioresolution of some basic DL-amino acids using a pharmaceutical industry waste as chiral impregnating reagent.

Direct enantiomeric resolution of DL-arginine, DL-histidine, DL-lysine, DL-valine and DL-leucine into their enantiomers was achieved by thin layer chromatography (TLC) on silica gel plates impregnated with optically pure (1R, 3R, 5R)-2-azabicyclo[3,3,0]octan-3-carbo-xylic acid (0.011 M) as a chiral selector which is a waste of a pharmaceutical industry. Different combinations of acetonitrile-methanol-water were found to be successful in resolving the DL-amino acids. The spots were detected by ninhydrin (0.2% in acetone) and the detection limit was 0.66 microg.

Amino Acids↗

PubMed1999

Sediment deposition rates on the continental margins of the eastern Arabian Sea using 210Pb, 137Cs and 14C.

Eight gravity cores from the active eastern continental margins of the Arabian Sea were dated using 210Pbxs, 137Cs and 14C. The short-term (< or = 100 years) sedimentation rates range from 0.06 to 0.66 cm/year where as the long-term (> or = 1000 years) ones using AMS 14C on planktonic foraminifera varied from 0.004 to 0.13 cm/year. For long-term chronology (< or = 50,000 years) AMS dating of well-cleaned planktonic foraminifera is most suited.

Animals↗

PubMed1999

Direct enantiomeric resolution of some 2-arylpropionic acids using (-)-brucine-impregnated thin-layer chromatography.

Direct enantioseparation of (+/-)-ibuprofen and (+/-)-flurbiprofen was achieved by two-dimensional thin-layer chromatography on silica gel plates impregnated with optically pure (-)-brucine as chiral selector. The solvent systems that were successful in resolving both the compounds were acetonitrile-methanol (16:3 v/v) for the first dimension and acetonitrile-methanol-water (16:3:0.4, v/v) for the second dimension. Iodine vapour was used for detection; the detection limit for both (+/-)-ibuprofen and (+/-)-flurbiprofen was 0.1 microgram.

Chromatography, Thin Layer↗

PubMed1998

Direct enantioseparation of some beta-adrenergic blocking agents using impregnated thin-layer chromatography.

The resolution of (+/-)-atenolol, (+/-)-propranolol and (+/-)-metoprolol into their enantiomers was achieved by TLC on silica-gel plates impregnated with optically pure L-lysine (0.5%) and L-arginine (0.5%) as the chiral selectors. In all cases, different combinations of acetonitrile-methanol solvent systems were found to be successful in resolving these compounds. Spots were detected using iodine vapour. The detection limit for both (+/-)-atenolol and (+/-)-propranolol was 2.6 microg and for (+/-)-metoprolol, it was 0.26 microg.

Adrenergic beta-Antagonists↗

PubMed1998

Estimation of group B streptococcus type III polysaccharide-specific antibody concentrations in human sera is antigen dependent.

The presence of immunoglobulin G (IgG) antibodies against group B streptococcus (GBS) type III polysaccharide (PS) has been correlated with protection against GBS disease. The GBS type III PS is structurally similar to the pneumococcal type 14 PS, differing only in the presence of sialic acid residues. Four different preparations of GBS type III PS were evaluated for their specificity in enzyme-linked immunosorbent assay (ELISA): free PS, free PS mixed with methylated human serum albumin (mHSA), PS conjugated to biotin and PS conjugated to human serum albumin. Three groups of human sera were used to evaluate these PS preparations: sera from recipients of a GBS PS vaccine, sera from women receiving a GBS type III PS-tetanus toxoid conjugate vaccine, and sera from nonimmunized healthy women of childbearing age. Estimated antibody concentrations were different depending on the PS preparation used. Using any of the four preparations, we were able to measure </=0.05 micrograms of IgG antibody to the GBS type III PS per ml. The specificity of the assay was determined by competitive inhibition with homologous and heterologous PS. The pneumococcal type 14 PS did not inhibit binding of antibody to the native GBS type III PS in sera from adults receiving the GBS PS vaccine or in sera from nonimmunized adults (except serum G9). The pneumococcal type 14 PS inhibited 50% in sera from recipients of GBS type III conjugate vaccine and in serum G9 when GBS type III PS conjugated to biotin or to HSA was used as antigen in ELISA. These data show that free GBS type III PS or PS mixed with mHSA is a sensitive and specific antigen for ELISA and that conjugation can alter the antigenic specificity of a PS.

Adult↗

PubMed1997

Antigenic characterization and analysis of the human immune response to outer membrane protein E of Branhamella catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa major OMP of Branhamella catarrhalis. Polyclonal antisera and four monoclonal antibodies (MAbs) to OMP E were generated to study its antigenic structure. All antibodies recognized epitopes in all 19 B. catarrhalis strains tested by immunoblot assays. By flow cytometry, it was determined that MAbs 1B3 and 9G10d recognized epitopes which are expressed on the surface of the intact bacterium, while MAbs IC11 and 7C10 recognized epitopes which were buried within the outer membrane. A competitive enzyme-linked immunosorbent assay showed that MAbs 1B3 and 9G10d recognize the same or closely related epitopes. Proteinase K treatment of whole bacterial cells revealed that MAbs 1B3 and 9G10d recognize a surface-exposed epitope located in the 17-kDa region towards the amino terminus of OMP E. The human serum and mucosal antibody responses to OMP E in adults with chronic bronchitis were studied. A majority of these patients had immunoglobulin A to OMP E in sputum supernatants. None of ten adults who experienced lower respiratory tract infections due to B. catarrhalis demonstrated a clear-cut rise in antibody titer to OMP E in serum or sputum supernatant. This study has demonstrated that OMP E has at least one surface-exposed epitope which is highly conserved among strains of B. catarrhalis and which is located in the amino-terminal 184 amino acids of the molecule.

Adult↗

PubMed1996

The amino acid sequence and structural class of the arachin subunit of molecular weight 29, 100A.

Arachin subunit (mol. wt. 29,100) from peanut of variety G-201 was separated and isolated, its purity and homogeneity were ascertained. The subunit was cleaved with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. The fragments obtained were separated and isolated by PAGE, gel filtration & ion exchange chromatography, these were subjected to amino acid analysis, and Edman degradation. The PTH amino acids obtained were identified by spectroscopy and TLC. The complete amino acid sequence of the subunit (226 residues) was established, and the structural class of arachin was predicted from the amino acid composition.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and characterization of outer membrane protein E of Moraxella (Branhamella) catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa protein of Moraxella (Branhamella) catarrhalis. It is a potential vaccine antigen because it is expressed on the surface of the bacterium and has antigenic determinants which are conserved among most strains of M. catarrhalis. To clone the gene encoding OMP E, an EMBL-3 genomic library of strain 25240 was screened with a family of degenerate oligonucleotides based on the amino-terminal protein sequence. The OMP E gene was identified in one of the six positive clones by Southern blot analysis. An open reading frame of 1,377 bp encoding a protein of 460 amino acids was identified. The calculated molecular mass of the mature protein of 436 amino acid residues was 47.03 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein product of the OMP E gene had a leader peptide of 25 amino acids and a signal peptidase 1 cleavage site similar to those of known OMPs of Escherichia coli. The transcription initiation site of the OMP E gene was mapped by primer extension to be 78 nucleotides upstream of the ATG start codon. Borderline homology was found to the FadL protein of E. coli (49.1% similarity and 25.6% identity), which is involved in the binding and transport of fatty acids. Analysis of restriction fragment length polymorphisms of the OMP E genes of 19 different strains of M. catarrhalis showed that the OMP E gene is highly conserved. The high degree of conservation of sequences of the OMP E genes of M. catarrhalis from diverse sources, along with earlier observations that the protein contains antigenic determinants on the bacterial surface, indicates that OMP E should be studied further as a potential vaccine antigen.

Amino Acid Sequence↗

PubMed1994

Glucocorticoids antagonize insulin's antiproteolytic action on skeletal muscle in humans.

Although chronic glucocorticoid elevations cause net skeletal muscle protein loss in man, the kinetic mechanisms responsible for this catabolic effect and the capacity of insulin to overcome it remain unclear. To examine this issue, we measured basal and insulin-stimulated rates of protein synthesis and breakdown in muscle using the phenylalanine forearm kinetic method in eight normal volunteers studied postabsorptively and after 4 days of dexamethasone treatment (8 mg/day). To avoid the confounding effects of systemic insulinization, local forearm insulin levels were raised by approximately 430 pmol/L using a 150-min brachial arterial infusion of insulin (0.251 pmol/kg.min). Postabsorptively, dexamethasone produced mild hyperglycemia (P < 0.003) and a 3-fold rise in plasma insulin (P < 0.001), but no change in forearm phenylalanine balance or kinetics. Before dexamethasone treatment, local hyperinsulinemia increased forearm glucose uptake 2.5-fold and caused a positive net balance of phenylalanine due to a marked 40% inhibition of proteolysis. After dexamethasone treatment, forearm glucose uptake was modestly reduced. However, forearm net phenylalanine balance remained negative due to a striking reduction in insulin's inhibitory effect on proteolysis. We conclude that 1) the effects of glucocorticoid on basal muscle protein turnover are minimized by compensatory hyperinsulinemia, and 2) glucocorticoids cause muscle resistance to insulin's antiproteolytic action.

Adolescent↗

PubMed1992

Pituitary response to growth hormone-releasing hormone in IDDM. Abnormal responses to insulin and hyperglycemia.

In poorly controlled insulin-dependent diabetes mellitus (IDDM), hyperglycemia fails to inhibit the pituitary response to growth hormone-releasing factor (GRF). To evaluate whether this derangement is reversed by a simultaneous elevation of circulating insulin, 0.3 micrograms/kg i.v. GRF 1-40 was administered to nine poorly controlled IDDM subjects (HbA1 greater than 11.1%) with and without concomitant infusion of insulin. In the absence of insulin, the poorly controlled IDDM subjects demonstrated a growth hormone response to GRF similar to that of nondiabetic subjects, despite marked hyperglycemia (approximately 16.8 mM). When insulin was infused into these same patients (insulin clamp) to produce combined hyperinsulinemia (528 +/- 90 pM) and hyperglycemia (16.5 +/- 1.98 mM), the GRF-induced growth hormone rise was markedly exaggerated (65 +/- 11 vs. 20 +/- 4 micrograms/L without insulin infusion, P less than 0.001). This enhancement of GRF-stimulated growth hormone release by insulin was strikingly attenuated (22 +/- 7 micrograms/L) in five well-controlled diabetic subjects studied under conditions of similar hyperinsulinemia (486 +/- 84 pM) and hyperglycemia (16.41 +/- 0.95 mM). In contrast, in nondiabetic subjects, acute hyperinsulinemia reduced the growth hormone response to GRF. We conclude that the failure of hyperglycemia to block the pituitary response to GRF in poorly controlled diabetes is not attributable to the lack of a coincident increase in circulating insulin. The paradoxical stimulatory effect of insulin on GRF-induced growth hormone release may contribute to the high spontaneous growth hormone levels characteristically seen in poorly controlled insulin-treated patients, and its attenuation after intensive insulin therapy may contribute to the reversal of growth hormone hypersecretion in well-controlled diabetic patients.

Adult↗

PubMed1990

Importance of enantiomeric purity and its control by thin-layer chromatography.

Methods for the direct resolution of enantiomers are important and are necessary for pharmaceutical and biomedical analysis, synthetic and mechanistic studies and various other fields. The present paper deals with the results of recent approaches, such as ligand exchange, ion exchange and steric interactions, providing direct resolution of enantiomers of a variety of compounds by thin-layer chromatography. General aspects of various methods for analysis of enantiomeric purity and resolution have been compared.

Amino Acids↗

PubMed1990

Reconstitution of rapeseed high molecular weight protein from isolated subunits.

The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide-gel electrophoresis and by gel filtration on Sephadex G-100. Reassociation by removing SDS by co-dialysis, against 10 mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N-terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.

Amino Acids↗

PubMed1990

Complete amino acid sequence of arachin subunit of molecular weight 21,000.

A subunit of molecular weight 21,000 from arachin, the major peanut protein, was isolated in pure form and primary structure was determined. The subunit was fragmented with CNBr, trypsin, and NBS; the fragments were separated and isolated by PAGE, gel filtration, Dowex treatment, and paper electrophoresis, and Edman degradation on each fragment, including the intact subunit, was performed. The PTH-amino acids thus obtained were identified by UV spectroscopy and TLC. The complete sequence of 176 residues was established by overlapping technique.

Amino Acid Sequence↗

PubMed1990

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein.

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.

Amino Acid Sequence↗